Previously, β-thalassemia, an inherited anemic disorder with iron overload caused by loss-of-function mutation of β-globin gene, has been reported to induce osteopenia and impaired whole-body calcium metabolism, but the pathogenesis of aberrant calcium homeostasis remains elusive. Herein, we investigated how β-thalassemia impaired intestinal calcium absorption and whether it could be restored by administration of 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] or hepcidin, the latter of which was liver-derived antagonist of intestinal iron absorption. The results showed that, in hemizygous β-globin knockout (BKO) mice, the duodenal calcium transport was lower than that in wild-type littermate, and severity was especially pronounced in female mice. Both active and passive duodenal calcium fluxes in BKO mice were found to be less than those in normal mice, which could be restored by 7-day 1,25(OH)₂D₃ treatment. Such the 1,25(OH)₂D₃-induced calcium transport was diminished by inhibitors of calcium transporters, e.g., L-type calcium channel, NCX1, PMCA_1b, as well as vesicular transport inhibitor. Interestingly, the duodenal calcium transport exhibited an inverse correlation with transepithelial iron transport, which was markedly enhanced in thalassemic mice. Thus, 3-day subcutaneous hepcidin injection and acute direct hepcidin exposure in Ussing chamber were capable of restoring the thalassemia-associated impairment of calcium transport; however, the positive effect of hepcidin on calcium transport was completely blocked by proteasome inhibitors, MG132 and bortezomib. In conclusion, 1,25(OH)₂D₃ impairment of calcium absorption. Our study has, therefore, shed light on the development of treatment strategy to rescue calcium dysregulation in β-thalassemia.